Fottea, Olomouc, 21(2): 259–271, 2021 259

DOI: 10.5507/fot.2021.011

Description of two new species of Nostoc from China based on the polyphasic
approach

Fangfang Cai1,2, Gongliang Yu2, Yuchen Liu 1, Yuanquan Sun1 & Renhui Li3*

1
Hubei Key Laboratory of Animal Nutrition and Feed Science, Hubei Collaborative Innovation Center for Animal
Nutrition and Feed Safety, Wuhan Polytechnic University, Wuhan 430023, China
2
Key Laboratory of Algal Biology, State Key Laboratory of Freshwater Ecology and Biotechnology of China,
Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
3
School of Life and Environmental Sciences, Wenzhou University, Zhejiang 325035, China; *Corresponding
author e–mail: renhui.li@wzu.edu.cn

Abstract: The present study described two new Nostoc species, Nostoc favosum (CHAB5709, CHAB5713, and
CHAB5714) and Nostoc mirabile (CHAB5756 and CHAB5784) based on the polyphasic approach combining
morphological, genetic and ecological characteristics. Five investigated strains were found to possess morpho-
logical features of the Nostoc genus. Results showed that the 16S rRNA gene sequences of these five strains
displayed ≥ 95%, and ≤ 98% similarity to the genus Nostoc. The 16S rRNA gene phylogenetic analysis inferred
using bayesian inference, maximum–likelihood and neighbour–joining methods placed these five strains on two
separate nodes away from other Nostoc species. The 16S–23S rRNA internal transcribed spacer (ITS) secondary
structure of two new species showed a unique pattern of D1–D1′, Box–B and V3 helix, which distinguished
them from other Nostoc species. And the two species were further established by percent dissimilarity of ITS
between another Nostoc species.

Key words: 16S rRNA gene, 16S–23S ITS, new species, morphology, polyphasic approach, taxonomy, Nostoc

Introduction for determination of cyanobacteria in nature) (Komárek

Cyanobacteria are a morphologically and ecologically
diverse group of photoprokaryotes found around the globe
(Graham et al. 2008). The traditionally classification
of cyanobacteria was mainly based on morphological
characteristics (Komárek 2003; Taton et al. 2003,
2006; Johansen & Casamatta 2005; Turicchia et al.
2009; Genuário et al. 2013). But many cyanobacterial
taxa are difficult to identify due to the lack of clear
morphological characteristics and the vague or overly
broad concept of modern genera. In recent decades,
however, additional applications of molecular data us-
ing 16S rRNA gene sequences have greatly improved
the classification and systematics of cyanobacteria
(Robertson et al. 2001; Komárek 2006; Fiore et al.
2001; Silva et al. 2014; Bravakos et al. 2016). This
progress in taxonomic research resulted in the descrip-
tion of many new cyanobacterial genera, which are
based on phylogenetic analyses. However, there still
exist several unsolved problems in genera established
after molecular sequencing (eg. identification of new
taxa by ecologists, who do not use molecular analyses
2020). In this situation, Komárek (2020) proposed that
modern classification must be applied consistently the
polyphasic approach with the phylogenetic classification
as the basis, but it is necessary to combine and add all
cytomorphological and ecological important data, and
adequate use of nomenclature prescriptions.
Genus Nostoc, differed from other heterocytous
cyanobacteria in that the trichomes with heterocytes and
akinetes are embedded in colonial common mucilage.
Despite being first reported in the 19th century with
type species Nostoc commune Vaucher ex Bornet et
Flahault (Bornet & Flahault 1888), its taxonomy
remains complicated and problematic. To date, over
100 species of Nostoc have been described worldwide
(Guiry & Guiry 2021), most of these species have
not yet been sequenced and have rarely been reported
in the literature. Also this taxonomically complicated
genus require nomenclatural revision (Komárek 2013).
Many of described Nostoc species may belong to other
genera, but is waiting for the taxonomical revision
(such as Nostoc ellipsosporum V, Nostoc spongiaeforme
Ind42, Nostoc verrucosum KU005, Nostoc insulare
260
SAG 54.79, Nostoc carneum IAM M–35 and Nostoc
piscinale CENA21 listed in NCBI database). In addi-
tion, Nostoc has repeatedly been proven to be geneti-
cally heterogeneous and polyphyletic (Hrouzek et al.
2005, 2013; Rajaniemi et al. 2005a; Papaefthimiou et
al. 2008; Lukešová et al. 2009; Silva et al. 2014). With
the advent of polyphasic approaches, some Nostoc–like
genera have been separated from Nostoc in an attempt
to make this genus less polyphyletic, including Mojavia
(Řeháková et al. 2007), Desmonostoc (Hrouzek et al.
2013), Halotia (Genuário et al. 2015), Aliinostoc (Bagchi
et al. 2017), Komarekiella (Hentschke et al. 2017) and
Desikacharya (Saraf et al. 2019). A subsequent recent
study that isolated several strains from rocky mountain
in China exhibit Nostoc–like appearance. The investi-
gation showed molecular distinction and the observed
cyanobacteria have been assigned with new names as
Minunostoc, Compactonostoc, Purpureonostoc and
Violetonostoc (Cai et al. 2019a, 2019b, 2020a, 2020b).
The study of terrestrial Nostoc–like cyanobac-
teria in China was limited till recent years, during a
broader survey on Chinese terrestrial Nostoc–like cya-
nobacteria, we have found and described several new
genera and species in the family Nostocaceae (Cai et
al. 2018, 2019a, 2019b, 2020a, 2020b). In this study,
we isolated morphologically distinct heterocytous cya-
nobacteria from seldomly sampled regions in China,
such as Baishan city in Jilin province and Shennongjia
Forestry District in Hubei province. A polyphasic ap-
proach was employed to intensively study five selected
strains (CHAB5709, CHAB5713, and CHAB5714,
CHAB5756 and CHAB5784). Morphological features
were observed and studied at different stage of their life
cycle. Morphologically, we concluded them as Nostoc,
and molecular analysis confirmed its position in Nostoc
sensu stricto. Moreover, phylogeny of 16S rRNA gene,
the secondary structure through sequencing of internally
transcribed spacer (ITS) between 16S rRNA and 23S
rRNA of the ribosomal genes, and percent dissimilarity
of ITS were also determined herein to estimate their exact
taxonomic status. Overall, the analyses of morphology
and molecular data revealed that these five strains did
not match any described species of Nostoc. These results
allowed us to describe two new Nostoc species, as Nostoc
favosum and Nostoc mirabile.
Materials and Methods
Sampling, isolation and culturing of strains. Samples used
in this study were isolated from two different localities of
China having different climatic and geographical conditions.
The strains CHAB5709, CHAB5713 and CHAB5714 were
isolated from the edge of a stream (42°51.92'N, 127°78.94'E)
in Baishan city, Jilin province, temperate climate in China.
The strains CHAB 5756 and CHAB 5784 were collected
from wet rocks in Shennongjia Forestry District (31°44.62'N,
110°30.92'E), located near the western border of Hubei province,
a subtropical monsoon climate in China. Unialgal filaments
Cai et al.: Description of two new species of Nostoc
from the cyanobacterial samples were isolated by lab–made
Pasteur pipette under microscope (Olympus C31, Japan) and
then cultured in screw capped tubes containing 6 ml of BG11
medium. All isolates were subsequently cultivated at 25 °C
under a 12:12 h (light: dark) cycle with a photon flux density
of 35 μmol.m–2.s–1 from white fluorescent lamps. The living
culture were maintained in the Chinese Harmful Algae Biology
(CHAB) culture collection of the Institute of Hydrobiology,
China. And dry materials of strains were obtained by freeze–
drying at –40 °C and stored at the Freshwater Algal Herbarium
(HBI), Institute of Hydrobiology, Chinese Academy of Science,
Wuhan, China.
Morphological characterization. All live cultures were ex-
amined with a Nikon Eclipse 80i microscope (Nikon, Tokyo,
Japan) using its differential interference contrast micros-
copy. Microphotographs were analyzed with Nikon software
NIS–Elements 3.2D (Nikon, Tokyo, Japan). Morphometric
characteristics included length and width of vegetative cells,
heterocytes and akinetes, as well as the size of macrocolonies
and microcolonies (n > 50) were described using the Nikon
Eclipse 80i microscope equipped with a Nikon DS–Ri1 digital
camera.
Molecular analysis. Total genomic DNA was isolated from
liquid cultures of unialgal cyanobacterial strains using Clarke’s
(Clarke 2009) method. The primers PA (Edwards et al. 1989)
and B23S (Gkelis et al. 2005) were used to amplify the 16S
rRNA gene. Primers 322 and 340 (Iteman et al. 2000) were
used to obtain the 16S–23S ITS region. The PCR reaction
contained 1 μl of genomic DNA (100 ng ΜL–1), 0.5 µl of
each primer (10 μmol.l–1), 8 μl of sterile water and 10 μl of 2×
PCR mix with Taq polymerase (Cat TSE001, Beijing Tsingke
BiotechCo., Ltd., Beijing, China), in a final volume of 20 µl.
PCR was performed in an MJ Mini Personal Thermal Cycler
(Bio–Rad, Hercules, California USA), and the PCR cycle
had initial denaturation at 94 °C for 3 min, followed by 34
cycles of 94 °C for 30 s, 58 °C for 30s (30 s at 55 °C for ITS),
72 °C for 1 min (30s for ITS), and a final 5 min elongation
step at 72 °C. The PCR amplification products were purified
using TSINGKE DNA Gel Extraction Kit (Cat GE0101–200,
Beijing Tsingke Biotech Co., Ltd., Beijing, China) and then
cloned into the pMDTM18–T vector (TaKaRa Bio Inc., Otsu,
Japan) using the procedure of Sambrook & Russell (2001).
All sequencing was carried out by the ABI 3730 Automated
Sequencer (PerkinElmer, Waltham, MA, USA). The 16S rRNA
and 16S–23S ITS gene sequences of the cyanobacterial strains
CHAB5709, 5713, 5714, 5756 and 5784, isolated in this study
were deposited in the NCBI GenBank database under accession
numbers: MW649141, MW649142, MW649809, MW649810,
MW649811, MW652791, MW652792, MW652793, MW652794,
and MW652795.
Phylogenetic analysis. 16S rRNA gene sequences obtained in
this study and those representing main groups of heterocytous
cyanobacteria retrieved from GenBank were used for phylo-
genetic analyses with in total 184 sequences. Sequences using
MAFFT v7.312 (Katoh & Standley 2013) with auto–selected
strategy FFT–NS–I (with default parameters) and visually
checked in mega v.7.0.14 (Kumar et al. 2016). The phylo-
genetic trees were constructed using neighbor–joining (NJ),
maximum likelihood (ML), and bayesian inference (BI). The
NJ analysis using Kimura–2 model upon default parameters
with 1000 bootstrap replicates were run via MEGA software
X (Kumar et al. 2018). The BI was calculated with MrBayes
Fottea, Olomouc, 21(2): 259–271, 2021
DOI: 10.5507/fot.2021.011
v3.2.6 (Ronquist et al. 2012) in the CIPRES Science Gateway
V.3.3 (Miller et al. 2015, http://www.phylo.org/), in the BI
analyses, two runs of eight Markov chains were executed for
8 million generations, sampling every 100 generations, with
25% of the sampled trees discarded as burn–in (the average
standard deviation of split frequencies is 0.003). In the ML
analyses, a total of 10000 bootstrap replicates were conducted
to evaluate the relative support for branches by performing
ultrafast bootstrap on IQ–TREE web server (Trifinopoulos et
al. 2016). All phylogenetic tree consensus files were visualized
in FigTree, v1.4.3 (Rambaut 2016) with Chroococcidiopsis
thermalis PCC7203 as the outgroup. Calculation of p–distance
with pairwise deletion of gaps was done with MEGA software
v.7.0.14 (Kumar et al. 2016) and used to calculate sequence
identity [100 × (1 – p)] for 16S rRNA data.
16S–23S secondary structure analysis. 16S–23S rRNA
secondary structures of D1–D1′, Box–B, and V3 helices were
determined using “RNAstructure”, ver. 5.6 (Mathews Lab
2013). The sequences containing both tRNAIle and tRNAAla
were used for all ITS analyses (except for Nostoc neudorfense
ARC8, which has no tRNAIle and tRNAAla). Percent dissimilarity
based on 16S–23S ITS was calculated based on p–distance.
Results
Nostoc favosum F. Cai et R. Li sp. nov. (Fig. 1)
Description: Colonies spherical, start yellow green, and
become to gray green. Filaments released by the colony
rupture, covered with or without sheath, and the fila-
ments densely entangled to form young colony. Within
the colony, the filaments segmented into several small
groups, and the small groups showing compartmentaliza-
tion of mucilage, later to form small spherical colony.
Consequently, the older colony consist of aggregations of
microcolonies, just like a beehive. Eventually the older
colony broken to release these small colonies. Sheath
thick, colorless. Vegetative cells short barrel shaped
to subspherical, or oblong, 2.7–3.1–3.8 μm long and
2.7–4.4–4.6 μm wide. Heterocytes subspherical, with
a diameter of 2.8–4.3–4.9 μm. Akinetes not observed
during years of cultivation.
Reference strains: The living culture were deposited in
Collection of Harmful Algae Biology (CHAB), Institute
of Hydrobiology, Chinese Academy of Sciences, Wuhan,
Hubei Province, China as strain CHAB5709, CHAB5713
and CHAB5714.
Type locality: Isolated from the edge of a stream in Jilin
province, China (42°51.92'N, 127°78.94'E).
Holotype here designated: Dry material of the strain
CHAB5709 was stored at the Freshwater Algal Herbarium
(HBI), Institute of Hydrobiology, Chinese Academy of
Science, Wuhan, China, as specimen No. JLCN201401.
Etymology: favosum = “favose”, refers to typical mor-
phological characteristics of this species.
Habitat: Free–living on edge of a stream in the soil.
261
Nostoc mirabile F. Cai et R. Li sp. nov. (Fig. 2)
Description: Thallus macroscopic in nature and in
culture. In culture, forming macroscopic masses on test
tube bottom in liquid media. On agar, macroscopic colo-
nies, as aggregation of spherical colonies with distinct
mucilage, which remain distinct throughout the whole
life cycle. Macrocolonies dark green. Filaments with no
sheath released from the colony, and later form distinct,
colorless sheath, and terminal heterocyte outside of the
sheath. Then the trichomes irregular, tightly entangled
in the sheath, to form young long filamentous colony,
the heterocytes appeared at the end of the colony. The
sheath outside of the colony, shrank to separate the
filamentous colony into smaller colonies. Later these
smaller colonies connected by heterocyte, and eventu-
ally formed spherical colonies. Vegetative cells elliptic
or spherical, 2.8–3.4–3.7 μm long and 3.0–3.5–3.9 μm
wide. Heterocytes larger than vegetative cells, short barrel
shaped that protrude from the population, subspherical
within the colony. 3.3–3.9–4.2 μm long, 3.6–4.1–4.8 μm
wide. Akinetes not observed during years of cultivation.
Reference strains: CHAB5756, CHAB5784, deposited in
Collection of Harmful Algae Biology (CHAB), Institute
of Hydrobiology, Chinese Academy of Sciences, Wuhan,
Hubei Province, China.
Type locality: Isolated from wet rocks in Shennongjia
Forestry District, Hubei province (31°44.62'N, 110°30.92'E).
Holotype here designated: Dry material of the strain
CHAB5784 was stored at the Freshwater Algal Herbarium
(HBI), Institute of Hydrobiology, Chinese Academy of
Science, Wuhan, China, as specimen No. HBCN201402.
Etymology: mirabile, referring to unique morphological
characteristics of this species.
Habitat: Free–living on wet rocky substrates.
Molecular and phylogenetic analysis
The 16S rRNA gene sequences of the studied strains
were obtained and evaluated with BLAST analyses in
NCBI. The evolutionary distance matrix based on 16S
rRNA gene showed that three Nostoc favosum strains
shared 99.3%–99.7% similarity with each other, but
they shared 96%–98% similarities with other Nostoc
species. Two Nostoc mirabile strains showed sequence
similarity of 99.4% to each other, while they showed
95%–97% to other Nostoc species (Table 1). The 16S
rRNA phylogenetic trees of ML, NJ and BI were con-
structed with 184 nucleotide sequences including the
studied strains, the nearly complete 16S rRNA gene
sequences after pairwise alignment was approximately
1166 bp. Bayesian tree showed that the studied Nostoc
strains, together with other Nostoc species, formed a
single cluster, and this unique cluster was supported by
NJ, ML, BI approaches with high bootstrap values of
100%, 100%, and 1.00, respectively (Fig. 3).
16S–23S ITS region
The percent dissimilarity among aligned ITS sequences
262 Cai et al.: Description of two new species of Nostoc

Fig. 1. Micrographs of Nostoc favosum under the light microscopy (LM): (A) free–living trichomes with or without heterocytes; (B–D) young
colony with densely entangled filaments; (E–F) older colony with a honeycomb–like structure is composed of a number of smaller spherical
microcolonies; (G–H) smaller spherical microcolonies released from the older colony. Scale bar 10 µm.
Fottea, Olomouc, 21(2): 259–271, 2021 263
DOI: 10.5507/fot.2021.011

Fig. 2. Micrographs of Nostoc mirabile under the light microscopy (LM): (A–C) filaments with internal and terminal heterocytes; (E, G) fila-
mentous young colony, showing heterocytes appeared at the end of the colony; (D, F) filamentous young colonies, connected by heterocytes;
(H) aggregation of spherical colonies with distinct mucilaginous. Scale bar 10 µm.
264 Cai et al.: Description of two new species of Nostoc

Fig. 3. Bayesian tree (BI) phylogenetic tree based on 16S rDNA sequences (1166bp) of the studied strains and other cyanobacterial strains.
Bootstrap values greater than 50% with NJ/ ML/Mrbayes methods are indicated on the tree, and the asterisks at the nodes mean 100. The novel
species are in bold font.

between our strains and the other Nostoc species for
which ITS sequence with both tRNA genes was avail-
able, varied from 7.5%–16.6%. Percent dissimilarity
of Nostoc favosum to other species is in the range of
11.4%–14.6%, and three Nostoc favosum strains shared
0.2% dissimilarity with each other. Percent dissimilarity
of Nostoc mirabile to other species is in the range from
11.2% to 16.6%, and two Nostoc mirabile strains shared
0% dissimilarity with each other (Table 2).
The secondary structures of conserved ITS
domains in Nostoc are highly similar (Figs 4–6), par-
ticularly in the D1–D1′ helices which were structurally
identical and had the similar patterns. Nostoc favosum
and Nostoc mirabile showed different nucleotides in the
Fottea, Olomouc, 21(2): 259–271, 2021 265
DOI: 10.5507/fot.2021.011

Fig. 4. Secondary structure of D1–D1’ helix: (A) Nostoc mirabile CHAB5784, CHAB 5756; (B) Nostoc favosum CHAB5709, CHAB5713,
CHAB5714; (C) Nostoc commune WY1–KK1; (D) Nostoc punctiforme PCC73102; (E) Nostoc desertorum CM1–VF14; (F) Nostoc indistingu-
endum CM1–VF10; (G) Nostoc lichenoides CNP–AK1; (H) Nostoc oromo ETH2.4.M5.

D1–D1’ helix in comparison with other Nostoc species
(Fig. 4A and 4B).
The Box–B helices of Nostoc were all very
similar at the basal part of the helix, but the sequences
and structure at the end are different, the eight Nostoc
species were divided into the eight types (Fig. 5). The
base stem of the Box–B helix of Nostoc mirabile (Fig.
5A) consisted of 5 bp helix, followed by a 3 : 3 base
bilateral bulge, and then further followed by a unpaired
nucleotide (A) on 3′ side, the terminal loop contained 5
bp bases (GAUGA). The base stem of Nostoc favosum
(Fig. 5B) consisted of 5 bp helix, and then followed by
a 3 : 3 base bilateral bulge, the terminal loop contained
6 bp bases (UUAAUU).
266 Cai et al.: Description of two new species of Nostoc

Fig. 5. Secondary structure of Box–B helix: (A) Nostoc mirabile CHAB5784, CHAB 5756; (B) Nostoc favosum CHAB5709, CHAB5713,
CHAB5714; (C) Nostoc commune WY1–KK1; (D) Nostoc punctiforme PCC73102; (E) Nostoc desertorum CM1–VF14; (F) Nostoc indistingu-
endum CM1–VF10; (G) Nostoc lichenoides CNP–AK1; (H) Nostoc oromo ETH2.4.M5.

Fig. 6. Secondary structure of V3 helix: (A) Nostoc mirabile CHAB5784, CHAB 5756; (B) Nostoc favosum CHAB5709, CHAB5713, CHAB5714;
(C) Nostoc commune WY1–KK1; (D) Nostoc punctiforme PCC73102; (E) Nostoc desertorum CM1–VF14; (F) Nostoc indistinguendum CM1–
VF10; (G) Nostoc oromo ETH2.4.M5; (H) Nostoc lichenoides CNP–AK1.

The V3 helices were similar only in the base part and
varied in structure and length (Fig. 6). The base stem
of Nostoc mirabile (Fig. 6A) consisted of 3 bp helix,
followed by a 2 : 2 base bilateral bulge, and then further
followed by a 1 : 3 base bilateral bulge, the terminal loop
contained 6 bp bases (UGUAAU). The base of the stem
of Nostoc favosum (Fig. 6B) consisted of 3 bp helix, and
then followed by two bilateral bulges, the terminal loop
contained a 6 bp nucleosides (CUUUAU).
Discussion
Due to the morphological plasticity, complex life cycle,
and huge diversity, the taxonomy of genus Nostoc be-
comes problematic (Mollenhauer et al. 1999; Komárek
et al. 2014; Singh et al. 2016). Molecular data showed
up that the genetic diversity of the genus exceeded its
morphological diversity, which clearly supported its
polyphyletic status (Rajaniemi et al. 2005a, 2005b;
Řeháková et al. 2007; Hrouzek et al. 2013; Genuário
Fottea, Olomouc, 21(2): 259–271, 2021 267
DOI: 10.5507/fot.2021.011

et al. 2015; Bagchi et al. 2017; Hentschke et al.

Table 1. Comparison of the 16S rRNA gene sequence similarity among the studied strains and its related taxa.
15.Nostoc edaphicum X
14.Nostoc cf. lichenoides JT1–VF3
13.Nostoc cf. indistinguendum F15–VF1
12.Nostoc lichenoides CNP–AK1
11.Nostoc indistinguenda CM1–VF10
10.Nostoc desertorum CM1–VF14
9.Nostoc oromo ETH. 2.4. M5
8.Nostoc neudorfense ARC8
7.Nostoc punctiforme PCC 73102
6.Nostoc commune WY1KK1
5.Nostoc mirabile CHAB5784
4.Nostoc mirabile CHAB5756
3.Nostoc favosum CHAB5714
2.Nostoc favosum CHAB5713
1.Nostoc favosum CHAB5709
2017). Recent efforts have been made to reevaluate
the genus by recognizing Nostoc sensu stricto, a
clade that includes the type species, Nostoc com-
mune, and excludes species that fall outside this
clade that bear the name Nostoc, but cannot be put
in that genus if monophyly must be achieved at the
genus level (Řeháková et al. 2007; Hrouzek et al.
2013; Genuário et al. 2015; Bagchi et al. 2017;
Hentschke et al. 2017; Saraf et al. 2019; Cai et
al. 2019a, 2019b, 2020a, 2020b).
Taxonomical studies have found that molecu-
lar evidences, morphological, biogeographical, and

0.975

0.977
0.976
0.965

0.971
0.975
0.977
0.975
0.972

0.993
0.999
0.98

0.96

0.97
ecological data are often consistent, and their com-

ID
1
bination can better distinguish species with similar
morphology. This is the essence of the polyphasic

0.976

0.978
0.977
0.966

0.972
0.976
0.978
0.976
0.973
0.971
0.994
0.98

0.96
approach, which has been followed when making

ID

2
decisions at the species level (Komárek 2018; Mai
et al. 2018). In this study, the species Nostoc favosum 0.975
0.978
0.975
0.976
0.967

0.967
0.975
0.977
0.973
0.972
0.96

0.97
and Nostoc mirabile were characterized on the basis

ID

3
of the polyphasic approach. The phylogenetic analyses
in this study, combined with the cut off values for
0.968
0.961

0.953
0.951

0.961
0.963
0.957
0.994
0.96
0.96

0.95
the genus and species delimitation, clearly indicated

ID

4
that investigated strains are member of the genus
Nostoc. In the 16S rRNA gene phylogenetic tree,
0.963

0.961
0.953
0.952
0.951
0.962
0.965
0.959
0.97

0.96

Nostoc favosum and Nostoc mirabile clustered within

ID

5
the Nostoc branch at a unique node. This indicated
that species Nostoc favosum and Nostoc mirabile
0.975
0.974
0.977
0.973
0.971
0.966
0.964
0.976
0.983

are new members of the genus Nostoc. The original ID

6
identity cut–off value recommended for separation
of bacterial species was 97.5% (Stackebrandt &
0.989
0.982
0.984
0.987

0.978
0.974

Goebel 1994). However, on the basis of extensive

0.98

0.99
ID

7
comparison of more bacterial strains, Stackebrandt
& Ebers (2006) recommended 98.7–99.0% of
0.986
0.979

0.983
0.973

0.972

16S rRNA gene sequence similarity as the species

0.98

0.97

ID

8
threshold. Other research proposed that 98.65% of
16S rRNA gene sequence similarity could be used
0.976
0.975
0.968
0.978

0.961

as a threshold to distinguish two species based on

0.96

ID

twofold cross–validation statistical test (Kim et al.

2014). The highest 16S rRNA gene sequence simi-
0.973
0.971

0.975
0.969

larity between the Nostoc favosum strains detected

0.97

ID

10

in this study and other Nostoc strains (Nostoc cf.

lichenoides JT1–VF3) is 98%. The highest 16S rRNA
0.972
0.966

gene sequence similarity of Nostoc mirabile strains

0.98
0.97
ID

11

with other Nostoc strains is 97%, which provided

the molecular evidence to support the recognition
0.987
0.988
0.975

of the two species we have named.

ID

12

Up to now, four species, Desikacharya

nostocoides (the type species of Desikacharya),
Desikacharya soli, Desikacharya thermotolerans and
0.978
0.98
ID

13

Desikacharya constricta have been reported under

the genus Desikacharya, which are considered certain
features such as constrictions along with prominent
0.982
ID

14

coiling (Kabirnataj et al. 2020). Kabirnataj et

al. (2020) discussed that Minunostoc cylindricum
should be a member of the genus Desikacharya,
in contrast, the Desikacharya species within the
lineage, were differentiated into two distinct clades
268 Cai et al.: Description of two new species of Nostoc

with high genetic divergences (less than 95% similarity

closest sister taxa.

Table 2. Percent dissimilarity based on 16S–23S ITS sequence for operons containing both tRNA genes among the studied strains and the
11.Nostoc oromo ETH.2.4.M5
10.Nostoc lichenoides CNP–AK1
9.Nostoc indistinguenda CM1–VF10
8.Nostoc desertorum CM1–VF14
7.Nostoc punctiforme PCC73102
6.Nostoc commune WY1KK1
5.Nostoc favosum CHAB5714
4.Nostoc favosum CHAB5713
3.Nostoc favosum CHAB5709
2.Nostoc mirabile CHAB5784
1.Nostoc mirabile CHAB5756
of 16S rRNA gene), and Desikacharya has been shown
to be polyphyletic in our analyses. The 16S rRNA gene
sequence of Desikacharya constricta had less than 95%
similarities to the type species Desikacharya nostocoides
and Desikacharya soli, however, it had 96.9% similarity
to Minunostoc cylindricum. The phylogenetic tree also
showed that the sequence of Desikacharya constricta
clustered within the Minunostoc clade. In addition, the
16S rRNA gene sequence similarities between Minunostoc
cylindricum and other Desikacharya strains (except for
Desikacharya constricta) were shown as less than 95%,
below the bacterial genus cut–off (Table S1). Thus, we

16.12
14.78
14.77
14.61
16.68
16.38
11.50
11.25
11.25
conclude that Minunostoc cylindricum should not belong

1
to the genus Desikacharya (below 95% sequences simi-
larities is a strong evidence for separation of genera),

16.12
14.78
14.77
14.61
16.68
16.38
11.50
11.25
11.25

2
and the taxonomic status of Desikacharya constricta
should be separated from the Desikacharya genus, and

14.40
14.10
13.79

12.84
13.86
11.47

0.20
0.20
belonged to the Minunostoc genus.

3
The 16S–23S ITS secondary structure analysis
has been demonstrated to be effective tool to differentiate
14.40
14.10
13.79

12.84
13.86
11.47

0.20

4
cyanobacteria species (Iteman et al. 2000; Řeháková
et al. 2007; Johansen et al. 2011; Bohunická et al.
14.65
14.10
13.79

12.84
11.47

14.11
2015; Berrendero–Gómez et al. 2016; Mareš 2018).

5
In this study, the secondary structures of D1–D1′, Box–B
and V3 helices were also analyzed, which enabled our
10.21
10.68

10.60
7.59

9.99

strains to be distinguished from other Nostoc species.

6
It is shown here that D1–D1′, Box–B and V3 helices of
8.64
9.63
9.55
8.78

Nostoc favosum and Nostoc mirabile strains were quite

7
different from other selected Nostoc species. In addition,
the percent dissimilarity within the 16S–23S ITS region is
10.79
10.50
8.16

8
increasingly useful for cyanobacterial species delimitation.
Percent dissimilarity within the same species has always
10.20
9.63

been less than 4.0%, with an average value below 2.0%,

9
percent dissimilarity between species is typically over
7.0% (Erwin & Thacker 2008; Osorio–Santos et al.
9.44

10

2014; Pietrasiak et al. 2014; Bohunická et al. 2015;

Mai et al. 2018; Mesfin et al. 2020). Nostoc favosum is
distinct from the other Nostoc species based on percent
dissimilarity of the ITS region, which is > 11.4%, and
Nostoc mirabile is > 11.2% (Table 2). And dissimilarity
in the ITS regions between these two new taxa is well
above the 4% level. This fits well within the criteria for
species differentiation based on percent dissimilarity of
> 4.0%. So, in this study, the percent dissimilarity in the
ITS regions strongly supported their separation into two
new Nostoc species.
Morphologically, the Nostoc mirabile strains
closely resembles Nostoc punctiforme (Hariot 1891,
p. 31), having filamentous colony stage, heterocytes
appeared at the end of the colony, and densely entangled
filaments in colony. Nostoc mirabile create macroscopic
colonies observable without microscopy, whereas Nostoc
punctiforme is only microscopic in nature. In addition,
the phylogenetic analysis of the 16S rDNA, 16S rRNA
gene similarity and the 16S–23S ITS, show that Nostoc
mirabile and Nostoc punctiforme are genetically not closely
related. The morphologically closet Nostoc species to
Nostoc favosum in the literature is Nostoc desertorum
Řeháková et Johansen (2007, p. 488, Figs 2, 27–33),
described from sandy desert soil in California. Nostoc
desertorum shares the characteristic of compartmen-
talization of colonial mucilage in older colony. Nostoc
favosum differs in that the filaments segmented into
several small groups in young colony, later many small
groups grow into spherical colony, surrounded by large
spherical sheath. Nostoc desertorum can form fairly flat
masses in culture, a feature never seen in Nostoc favosum.
Ecologically, Nostoc favosum strains were described
from the edge of a stream, humid climate very differ-
ent from the sandy desert soil habitat in which Nostoc
desertorum was found.
In summary, two new species of Nostoc are
separated based on a combination of the 16S rRNA
gene–based phylogeny, 16S rRNA gene threshold of
98.65%, the ITS secondary structures, 16S–23S ITS
percent similarity, as well as morphological characteristics
Fottea, Olomouc, 21(2): 259–271, 2021
DOI: 10.5507/fot.2021.011
and ecological parameters. In China, the investigation
and researches on Nostoc–like cyanobacteria from all
kinds of environmental conditions have been largely
performed, and several new genera and species have
been found and published in the family Nostocaceae.
The investigation of less studied regions and habitats in
China could reveal the diversity of family Nostocaceae
and bring new inside into the diversity of cyanobacteria
on the world.
Acknowledgments
This research was supported by the National Natural Science Foundation
of China (32000166) and Science and Technology Research Program
of Hubei Provincial Department of Education (Q20201609).
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Supplementary material
The following supplementary material is available for this article:
Table S1. Comparison of the 16S rRNA gene sequence
similarity among Desikacharya and Minunostoc.
This material is available as part of the online article (http://
fottea.czechphycology.cz/contents)
© Czech Phycological Society (2021)
Received March 2, 2021
Accepted May 11, 2021